PDZ domain-dependent regulation of NHE3 protein by both internal Class II and C-terminal Class i PDZ-binding motifs

Boyoung Cha, Jianbo Yang, Varsha Singh, Nicholas Zachos, Rafiquel Sarker, Tian E. Chen, Molee Chakraborty, Chung Ming Tse, Mark Donowitz

Research output: Contribution to journalArticle

Abstract

NHE3 directly binds Na+/H+ exchanger regulatory factor (NHERF) family scaffolding proteins that are required for many aspects of NHE3 regulation. The NHERFs bind both to an internal region (amino acids 586-660) of the NHE3 C terminus and to theNHE3C-terminal four amino acids. The internal NHERFbinding region contains both putative Class I (-592SAV-) and Class II (-595CLDM-) PDZ-binding motifs (PBMs). Point mutagenesis showed that only the Class II motif contributes to NHERF binding. In this study, the roles in regulation of NHE3 activity of these two PBMs were investigated, revealing the following findings. 1) Interaction occurred between these binding sites because mutation of either removed nearly all NHERF binding. 2) Mutations in either significantly reduced basal NHE3 activity. Total and percent plasma membrane (PM) NHE3 protein expression was reduced in the C-terminal but not in the internal PBD mutation. 3) cGMP- and Ca2+-mediated inhibition of NHE3 was impaired in both the internal and the C-terminalPBMmutations. 4) There was a significant reduction in half-life of the PM pool of NHE3 in only the internal PBM mutation but no change in total NHE3 half-life in either. 5) There were some differences in NHE3-associating proteins in the two PBM mutations. In conclusion, NHE3 binds to NHERF proteins via both an internal Class II PBM and C-terminal Class I PBM, which interact. The former determinesNHE3stability in the PM, and the latter determines total expression and percent PM expression.

Original languageEnglish (US)
Pages (from-to)8279-8290
Number of pages12
JournalJournal of Biological Chemistry
Volume292
Issue number20
DOIs
StatePublished - May 19 2017

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PDZ Domains
Cell membranes
Mutation
Cell Membrane
Proteins
Half-Life
Amino Acids
Mutagenesis
Blood Proteins
Membrane Proteins
Binding Sites
sodium-hydrogen exchanger regulatory factor

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

PDZ domain-dependent regulation of NHE3 protein by both internal Class II and C-terminal Class i PDZ-binding motifs. / Cha, Boyoung; Yang, Jianbo; Singh, Varsha; Zachos, Nicholas; Sarker, Rafiquel; Chen, Tian E.; Chakraborty, Molee; Tse, Chung Ming; Donowitz, Mark.

In: Journal of Biological Chemistry, Vol. 292, No. 20, 19.05.2017, p. 8279-8290.

Research output: Contribution to journalArticle

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abstract = "NHE3 directly binds Na+/H+ exchanger regulatory factor (NHERF) family scaffolding proteins that are required for many aspects of NHE3 regulation. The NHERFs bind both to an internal region (amino acids 586-660) of the NHE3 C terminus and to theNHE3C-terminal four amino acids. The internal NHERFbinding region contains both putative Class I (-592SAV-) and Class II (-595CLDM-) PDZ-binding motifs (PBMs). Point mutagenesis showed that only the Class II motif contributes to NHERF binding. In this study, the roles in regulation of NHE3 activity of these two PBMs were investigated, revealing the following findings. 1) Interaction occurred between these binding sites because mutation of either removed nearly all NHERF binding. 2) Mutations in either significantly reduced basal NHE3 activity. Total and percent plasma membrane (PM) NHE3 protein expression was reduced in the C-terminal but not in the internal PBD mutation. 3) cGMP- and Ca2+-mediated inhibition of NHE3 was impaired in both the internal and the C-terminalPBMmutations. 4) There was a significant reduction in half-life of the PM pool of NHE3 in only the internal PBM mutation but no change in total NHE3 half-life in either. 5) There were some differences in NHE3-associating proteins in the two PBM mutations. In conclusion, NHE3 binds to NHERF proteins via both an internal Class II PBM and C-terminal Class I PBM, which interact. The former determinesNHE3stability in the PM, and the latter determines total expression and percent PM expression.",
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T1 - PDZ domain-dependent regulation of NHE3 protein by both internal Class II and C-terminal Class i PDZ-binding motifs

AU - Cha, Boyoung

AU - Yang, Jianbo

AU - Singh, Varsha

AU - Zachos, Nicholas

AU - Sarker, Rafiquel

AU - Chen, Tian E.

AU - Chakraborty, Molee

AU - Tse, Chung Ming

AU - Donowitz, Mark

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AB - NHE3 directly binds Na+/H+ exchanger regulatory factor (NHERF) family scaffolding proteins that are required for many aspects of NHE3 regulation. The NHERFs bind both to an internal region (amino acids 586-660) of the NHE3 C terminus and to theNHE3C-terminal four amino acids. The internal NHERFbinding region contains both putative Class I (-592SAV-) and Class II (-595CLDM-) PDZ-binding motifs (PBMs). Point mutagenesis showed that only the Class II motif contributes to NHERF binding. In this study, the roles in regulation of NHE3 activity of these two PBMs were investigated, revealing the following findings. 1) Interaction occurred between these binding sites because mutation of either removed nearly all NHERF binding. 2) Mutations in either significantly reduced basal NHE3 activity. Total and percent plasma membrane (PM) NHE3 protein expression was reduced in the C-terminal but not in the internal PBD mutation. 3) cGMP- and Ca2+-mediated inhibition of NHE3 was impaired in both the internal and the C-terminalPBMmutations. 4) There was a significant reduction in half-life of the PM pool of NHE3 in only the internal PBM mutation but no change in total NHE3 half-life in either. 5) There were some differences in NHE3-associating proteins in the two PBM mutations. In conclusion, NHE3 binds to NHERF proteins via both an internal Class II PBM and C-terminal Class I PBM, which interact. The former determinesNHE3stability in the PM, and the latter determines total expression and percent PM expression.

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